1. The Field of the Invention
The present invention relates to a system and method for the separation of biological and chemical materials which are in a liquid solution. More particularly, the present invention relates to a micromachined system for separation of molecules and particulate materials which utilizes electrical field-flow fractionation techniques.
2. The Relevant Technology
Field flow fractionation techniques are commonly used to perform separation and analysis of molecules and large molecular complexes which are in solution form. In theory, several different mechanisms can be employed to separate particles of differing varieties from a fluid stream; an example being separation by differences in mass or electrochemical potential through the application of an appropriate "driving" force. This driving force can be used to induce a displacement of a particular species in a certain direction such as towards what is commonly termed the "accumulation wall." From a practical point of view, a particularly convenient means of separation can be achieved through the application of an electric field, since electric fields are easily applied, controlled, and monitored in a typical laboratory setting. Therefore, the electric field is generally accepted as being one of the preferred methods to achieve separation of particles in solution form when accurate quantitative results are required.
In many applications, and particularly in biotechnology, there is a great demand for fast separation systems which have an adequate degree of resolution. Various methods have been proposed to achieve the separation of particles such as polymers, cells, and viruses. The majority of these methods, however, have various difficulties which include being too slow, too complicated, low in throughput, low in resolution, and too expensive to be practical for commercial and industrial applications.
Other techniques exist for the separation of both small molecules and also larger complexes of molecules, such as organelles and cells, which are in solute form. For example, molecular separations can be performed with high yield by ion exchange or reverse phase chromatography, which are methods that use chemicals to achieve separation. Chemical separations are observed, however, to have the undesirable effect of denaturing proteins, and are therefore unsuitable for certain applications. Centrifugation can be used to separate cells and organelles, although this technique often fails to resolve different types of cells due to similarities in cell densities. Molecular separation can also be done by electrophoresis, which separates samples by variation in molecular size. Typical electrophoresis systems require very high field strengths, which can result in the unwanted formation of electrolysis by-products in the vicinity of the electrodes. Conventional electrophoresis systems also employ a vertically oriented channel through which the sample containing the fluid must flow, which can result in the disadvantage of distortion in laminar flow conditions due to the effects of gravity.
In contrast with electrophoresis separation systems, electric field flow fractionation (EFFF) techniques utilize an electric field which is applied perpendicular to the direction of flow, and achieve separation by distinguishing amongst particles which flow at different velocities through a thin, horizontally oriented (with respect to the gravitational field) channel. The difference in velocities of various particles in the liquid arises from differences in the size and charge of each particle, quantities which are characterized together in a parameter which has been termed as the ".zeta. potential." The .zeta. potential of a particle is a useful parameter in that it may be considered to be a measure of its effective charge.
In EFFF techniques, the electrodes are placed on the top and bottom of the thin channel, allowing for the application of the electric field in a direction perpendicular to the flow. The EFFF process is based on a distinction amongst the differing velocities of the particles, and the forcing of particles with higher .zeta. potential towards the accumulation wall of the channel, while particles with lower .zeta. potential remain in the middle of the stream. As the various particles flow through the channel under the influence of the electric field, particles of differing .zeta. potentials and sizes will flow at different rates. The final equilibrium position of a species is determined by the combined effect of parameters such as the magnitude of the applied voltage and its polarity, the channel dimensions, and the viscosity of the flow medium. Various species of different sizes and different charges will thus be separated in accordance with their .zeta. potentials along different locations in the flow channel.
For the fluid velocities and channel dimensions utilized in EFFF systems, the Reynolds number is less than one. For this case, the flow in such a channel may be considered to be laminar. Laminar flow conditions imply that the velocity of the fluid flow is greater near the center of the channel and that the velocity is slowest near the walls of the channel, and hence may be well approximated by a substantially parabolic profile with respect to the velocity as a function of lateral position in the channel.
The EFFF techniques have all of the advantages of electrophoretic separation systems, but in addition, can satisfactorily perform separation of cells, large molecules, colloids, emulsions, and delicate structures such as liposomes, which cannot be accomplished by conventional electrophoretic systems. Possible applications where EFFF systems can supersede or complement electrophoretic separation systems include sample purification, cell separations, characterizations of emulsions, separations of particulates for intravenous drug administration, diagnostic tests for specific molecules in colloidal suspensions, and research into various aspects of .zeta. potentials.
Various field flow fractionation systems have been developed in the past, such as that disclosed in U.S. Pat. No. 4,737,268 to Giddings. The system in Giddings' patent specifies a thin channel in which the fluid containing the particles to be separated flows in a laminar fashion under the application of a field or field gradient applied transversely to the direction of flow, in order to separate the various particles into different stream laminae. The flow rate is adjusted in such a way as to ensure laminar flow conditions and under the influence of the field or field gradient, the different species of particles will approach different transverse equilibrium distributions. The system also includes a splitting means to separate and recover the substreams which contain the various types of particles.
Another innovation in the area of electric field flow fractionation is disclosed in U.S. Pat. No. 5,240,618 to Caldwell et al., which describes an apparatus including a thin flow channel having top and bottom walls that are formed such that the inner surfaces thereof are made of an electrically conductive material to function as electrodes. The carrier fluid flowing through the channel can be water containing a red-ox couple such as quinone/hydroquinone to reduce polarization effects.
The last several years have seen tremendous development in the area of fabrication techniques for small (on the order of a micron) sized separation systems. This has been made possible by the progress in techniques utilized in the everyday fabrication of integrated circuits and semiconductor microsensors. The fabrication technologies utilized therein are commonly referred to as "micromachining" methods. A number of electrophoresis systems have been previously constructed by micromachining techniques, which typically include steps such as chemical etching of semiconductor wafers, thin film deposition, lithographic patterning, etc.
In U.S. Pat. No. 4,908,112 to Pace, a micromachined analytical separation device is disclosed in which a capillary sized conduit is formed by a channel in a semiconductor device, with the channel closed by a glass plate. A series of electrodes are positioned in the channel which are used to activate the motion of liquids flowing through the conduit.
Prior micromachined EFFF systems have typically utilized materials including titanium, gold, and certain plastics which have previously been demonstrated to be biocompatible. However, the plastics which have been hitherto implemented have been the source of complications in the fabrication steps.
It would therefore be of substantial interest to develop a system which is capable of separating small samples with enhanced resolution, and which overcomes the difficulties associated with prior systems.